Identification and conformer analysis of a novel redox-active motif, Pro-Ala-Ser-Cys-Cys-Ser, in Drosophila thioredoxin reductase by semiempirical molecular orbital calculation

Mammalian thioredoxin reductases (TrxRs) contain selenium as selenocysteine (Sec) in the C-terminal redox center -Gly-Cys-Sec-Gly-OH to reduce Trx and other substrates; a Sec-to-Cys substitution in mammalian TrxR yields an almost inactive enzyme. The corresponding tetrapeptide sequence in Drosophila melanogaster TrxR (Dm-TrxR), -Ser-Cys-Cys-Ser-OH, endows the orthologous enzyme with a catalytic competence similar to mammalian selenoenzymes, but implementation of the Ser-containing tetrapeptide sequence SCCS into the mammalian enzyme does not restore the activity of the Sec-to-Cys mutant form (turnover number <2/min). MOPAC calculation suggested that the C-terminal hexapeptide Pro-Ala-Ser-Cys-Cys-Ser-OH functions as a redox center that alleviates the necessity for selenium in Dm-TrxR, and a mutant form of human lung TrxR that mimics this hexapeptide sequence showed improved catalytic turnover (17.4/min for DTNB and 13.2/min for E. coli trx) compared to the Sec-to-Cys mutant. MOPAC calculation also suggested that the dominant form of the Pro-containing hexapeptide is a C+ conformation, which perhaps has a catalytic advantage in facile reduction of the intramolecular disulfide bond between Cys497 and Cys498 by the N-terminal redox center in the neighboring subunit.     

Roles of N-terminal active cysteines and C-terminal cysteine-selenocysteine in the catalytic mechanism of mammalian thioredoxin reductase

Mammalian thioredoxin reductase [EC 1.6.4.5], a homodimeric flavoprotein, has a marked similarity to glutathione reductase. The two cysteines in the N-terminal FAD domain (-Cys59-x-x-x-x-Cys64-) and histidine (His472) are conserved between them at corresponding positions, but the mammalian thioredoxin reductase contains a C-terminal extension of selenocysteine (Sec or U) at the penultimate position and a preceding cysteine (-Gly-Cys497-Sec498-Gly). Introduction of mutations into the cloned rat thioredoxin reductase gene revealed that residues Cys59, Cys64, His472, Cys497, and Sec498, as well as the sequence of Cys497 and Sec498 were essential for thioredoxin-reducing activity. To analyze the catalytic mechanism of the mammalian thioredoxin reductase, the wild-type, U498C, U498S, C59S, and C64S were overproduced in a baculovirus/insect cell system and purified. The wild-type thioredoxin reductase produced in this system, designated as WT, was found to lack the Sec residue and to terminate at Cys497. A Sec-containing thioredoxin reductase, which was purified from COS-1 cells transfected with the wild-type cDNA, was designated as SecWT and was used as an authentic enzyme. Among mutant enzymes, only U498C retained a slight thioredoxin-reducing activity at about three orders magnitude lower than SecWT. WT, U498C, and U498S showed some 5,5'-dithiobis(2-nitrobenzoic acid)-reducing activity and transhydrogenase activity, and C59S and C64S had substantially no such activities. These data and spectral analyses of these enzymes suggest that Cys59 and Cys64 at the N-terminus, in conjunction with His472, function as primary acceptors for electrons from NADPH via FAD, and that the electrons are then transferred to Cys497-Sec498 at the C-terminus for the reduction of oxidized thioredoxin in the mammalian thioredoxin reductase.     

Studies of an active site mutant of the selenoprotein thioredoxin reductase: the Ser-Cys-Cys-Ser motif of the insect orthologue is not sufficient to replace the Cys-Sec dyad in the mammalian enzyme

We have mutated the redox active C-terminal motif, Gly-Cys-Sec-Gly, of the mammalian selenoprotein thioredoxin reductase (TrxR) to mimic the C-terminal Ser-Cys-Cys-Ser motif of the non-selenoprotein orthologue of Drosophila melanogaster (DmTrxR). The activity of DmTrxR is almost equal to that of mammalian TrxR, which is surprising, because Cys mutants of selenoproteins are normally 1-2 orders of magnitude less active than their selenocysteine (Sec) containing counterparts. It was shown earlier that the flanking Ser residues were important for activating the Cys residues in DmTrxR (Gromer, et.al. (2003) PNAS 100, 12618-12623). However, the "Drosophila mimic" mutant of the mammalian enzyme studied herein had <0.5% activity compared to wild-type. Rapid kinetic studies revealed that all of the redox centers of the mutant were active, but that the C-terminal dithiols were not effective reductants of thioredoxin. The charge-transfer complex of the two-electron reduced enzyme slowly disappeared as the N-terminal dithiols reduced the C-terminal disulfide. In wild-type enzyme, the selenenylsulfide is more difficult to reduce and the charge-transfer complex is more stable. These findings suggest that features in addition to the flanking Ser residues are important for facilitating the high activity of the insect enzyme and that the corresponding features are absent in mammalian TrxR.     

Metabolism of selenium compounds catalyzed by the mammalian selenoprotein thioredoxin reductase

The mammalian thioredoxin reductases (TrxR) are selenoproteins with a catalytic selenocysteine residue which in the oxidized enzyme forms a selenenylsulfide and in the reduced enzyme is present as a selenolthiol. Selenium compounds such as selenite, selenodiglutathione and selenocystine are substrates for the enzyme with low K_m-values and the enzyme is implicated in reductive assimilation of selenium by generating selenide for selenoprotein synthesis. Redox cycling of reduced metabolites of these selenium compounds including selenide with oxygen via TrxR and reduced thioredoxin (Trx) will oxidize NADPH and produce reactive oxygen species inducing cell death at high concentrations explaining selenite toxicity. There is no free pool of selenocysteine since this would be toxic in an oxygen environment by redox cycling via thioredoxin systems. The importance of selenium compounds and TrxR in cancer and cardiovascular diseases both for prevention and treatment is discussed. A selenazol drug like ebselen is a direct substrate for mammalian TrxR and dithiol Trx and ebselen selenol is readily reoxidized by hydrogen peroxide and lipid hydroperoxides, acting as an anti-oxidant and anti-inflammatory drug.     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase activity

The substrate spectrum of human thioredoxin reductase (hTrxR) is attributed to its C-terminal extension of 16 amino acids carrying a selenocysteine residue. The concept of an evolutionary link between thioredoxin reductase and glutathione reductase (GR) is presently discussed and supported by the fact that almost all residues at catalytic and substrate recognition sites are identical. Here, we addressed the question if a deletion of the C-terminal part of TrxR leads to recognition of glutathione disulfide (GSSG), the substrate of GR. We introduced mutations at the putative substrate binding site to enhance GSSG binding and turnover. However, none of these enzyme species accepted GSSG as substrate better than the full length cysteine mutant of TrxR, excluding a role of the C-terminal extension in preventing GSSG binding. Furthermore, we show that GSSG binding at the N-terminal active site of TrxR is electrostatically disfavoured.     

The mechanism of high Mr thioredoxin reductase from Drosophila melanogaster

Drosophila melanogaster thioredoxin reductase-1 (DmTrxR-1) is a key flavoenzyme in dipteran insects, where it substitutes for glutathione reductase. DmTrxR-1 belongs to the family of dimeric, high Mr thioredoxin reductases, which catalyze reduction of thioredoxin by NADPH. Thioredoxin reductase has an N-terminal redox-active disulfide (Cys57-Cys62) adjacent to the flavin and a redox-active C-terminal cysteine pair (Cys489'-Cys490' in the other subunit) that transfer electrons from Cys57-Cys62 to the substrate thioredoxin. Cys489'-Cys490' functions similarly to Cys495-Sec496 (Sec = selenocysteine) and Cys535-XXXX-Cys540 in human and parasite Plasmodium falciparum enzymes, but a catalytic redox center formed by adjacent Cys residues, as observed in DmTrxR-1, is unprecedented. Our data show, for the first time in a high Mr TrxR, that DmTrxR-1 oscillates between the 2-electron reduced state, EH2, and the 4-electron state, EH4, in catalysis, after the initial priming reduction of the oxidized enzyme (Eox) to EH2. The reductive half-reaction consumes 2 eq of NADPH in two observable steps to produce EH4. The first equivalent yields a FADH--NADP+ charge-transfer complex that reduces the adjacent disulfide to form a thiolate-flavin charge-transfer complex. EH4 reacts with thioredoxin rapidly to produce EH2. In contrast, Eox formation is slow and incomplete; thus, EH2 of wild-type cannot reduce thioredoxin at catalytically competent rates. Mutants lacking the C-terminal redox center, C489S, C490S, and C489S/C490S, are incapable of reducing thioredoxin and can only be reduced to EH2 forms. Additional data suggest that Cys57 attacks Cys490' in the interchange reaction between the N-terminal dithiol and the C-terminal disulfide.     

The role of electrostatics in TrxR electron transfer mechanism: A computational approach

Thioredoxin reductase (TrxR) is an important enzyme in the control of the intracellular reduced redox environment. It transfers electrons from NADPH to several molecules, including its natural partner, thioredoxin. Although there is a generally accepted model describing how the electrons are transferred along TrxR, which involves a flexible arm working as a “shuttle,” the molecular details of such mechanism are not completely understood. In this work, we use molecular dynamics simulations with Poisson–Boltzmann/Monte Carlo pKa calculations to investigate the role of electrostatics in the electron transfer mechanism. We observed that the combination of redox/protonation states of the N‐terminal (FAD and Cys59/64) and C‐terminal (Cys497/Selenocysteine498) redox centers defines the preferred relative positions and allows for the flexible arm to work as the desired “shuttle.” Changing the redox/ionization states of those key players, leads to electrostatic triggers pushing the arm into the pocket when oxidized, and pulling it out, once it has been reduced. The calculated pKa values for Cys497 and Selenocysteine498 are 9.7 and 5.8, respectively, confirming that the selenocysteine is indeed deprotonated at physiological pH. This can be an important advantage in terms of reactivity (thiolate/selenolate are more nucleophilic than thiol/selenol) and ability to work as an electrostatic trigger (the “shuttle” mechanism) and may be the reason why TrxR uses selenium instead of sulfur. Proteins 2016; 84:1836–1843. © 2016 Wiley Periodicals, Inc.     

Overexpression of wild type and SeCys/Cys mutant of human thioredoxin reductase in E. coli: the role of selenocysteine in the catalytic activity

In contrast to Escherichia coli and yeast thioredoxin reductases, the human placental enzyme contains an additional redox center consisting of a cysteine-selenocysteine pair that precedes the C-terminal glycine residue. This reactive selenocysteine-containing center imbues the enzyme with its unusually wide substrate specificity. For expression of the human gene in E. coli, the sequence corresponding to the SECIS element required for selenocysteine insertion in E. coli formate dehydrogenase H was inserted downstream of the TGA codon in the human thioredoxin reductase gene. Omission of this SECIS element from another construct resulted in termination at UGA. Change of the TGA codon to TGT gave a mutant enzyme form in which selenocysteine was replaced with cysteine. The three gene products were purified using a standard isolation protocol. Binding properties of the three proteins to the affinity resins used for purification and to NADPH were similar. The three proteins occurred as dimers in the native state and exhibited characteristic thiolate-flavin charge transfer spectra upon reduction. With DTNB as substrate, compared to native rat liver thioredoxin reductase, catalytic activities were 16% for the recombinant wild type enzyme, about 5% for the cysteine mutant enzyme, and negligible for the truncated enzyme form.     

Seleno-independent glutathione peroxidases. More than simple antioxidant scavengers

Glutathione peroxidases (GPXs, EC 1.11.1.9) were first discovered in mammals as key enzymes involved in scavenging of activated oxygen species (AOS). Their efficient antioxidant activity depends on the presence of the rare amino-acid residue selenocysteine (SeCys) at the catalytic site. Nonselenium GPX-like proteins (NS-GPXs) with a Cys residue instead of SeCys have also been found in most organisms. As SeCys is important for GPX activity, the function of the NS-GPX can be questioned. Here, we highlight the evolutionary link between NS-GPX and seleno-GPX, particularly the evolution of the SeCys incorporation system. We then discuss what is known about the enzymatic activity and physiological functions of NS-GPX. Biochemical studies have shown that NS-GPXs are not true GPXs; notably they reduce AOS using reducing substrates other than glutathione, such as thioredoxin. We provide evidence that, in addition to their inefficient scavenging action, NS-GPXs act as AOS sensors in various signal-transduction pathways.     

Oxidation of thioredoxin reductase in HeLa cells stimulated with tumor necrosis factor-alpha

Stimulation of cells with tumor necrosis factor-alpha (TNF-alpha) results in the increase in generation of H(2)O(2) in mitochondria that leads to apoptosis. The effect of H(2)O(2) produced by TNF-alpha on the redox status of selenocysteine (SeCys) residue essential for mitochondrial thioredoxin reductase (TrxR2) was investigated in HeLa cells. TNF-alpha caused accumulation of oxidized TrxR2 with a thioselenide bond. The conditional induction of SeCys-deficient TrxR2 resulted in the increased production of H(2)O(2) and apoptosis. These results suggest that the SeCys residue of TrxR2 plays a critical role in cell survival by serving as an electron donor for Trx-II and subsequent peroxiredoxin-III, which is a primary line of defense against H(2)O(2) in mitochondria.     

Thioredoxin reductase as a pathophysiological factor and drug target.

Human cytosolic thioredoxin reductase (TrxR), a homodimeric protein containing 1 selenocysteine and 1 FAD per subunit of 55 kDa, catalyses the NADPH-dependent reduction of thioredoxin disulfide and of numerous other oxidized cell constituents. As a general reducing enzyme with little substrate specificity, it also contributes to redox homeostasis and is involved in prevention, intervention and repair of damage caused by H2O2-based oxidative stress. Being a selenite-reducing enzyme as well as a selenol-containing enzyme, human TrxR plays a central role in selenium (patho)physiology. Both dietary selenium deficiency and selenium oversupplementation, a lifestyle phenomenon of our time, appear to interfere with the activity of TrxR. Selenocysteine 496 of human TrxR is a major target of the anti-rheumatic gold-containing drug auranofin, the formal Ki for the stoichiometric inhibition being 4 nM. The hypothesis that TrxR and extracellular thioredoxin play a pathophysiologic role in chronic diseases such as rheumatoid arthritis, Sj?gren's syndrom, AIDS, and certain malignancies, is substantiated by biochemical, virological, and clinical evidence. Reduced thioredoxin acts as an autocrine growth factor in various tumour diseases, as a chemoattractant, and it synergises with interleukins 1 and 2. The effects of anti-tumour drugs such as carmustine and cisplatin can be explained in part by the inhibition of TrxR. Consistently, high levels of the enzyme can support drug resistance. TrxRs from different organisms such as Escherichia coli, Mycobacterium leprae, Plasmodium falciparum, Drosophila melanogaster, and man show a surprising diversity in their chemical mechanism of thioredoxin reduction. This is the basis for attempts to develop specific TrxR inhibitors as drugs against bacterial infections like leprosy and parasitic diseases like amebiasis and malaria.     

Catalytic activity of selenomethionine in removing amino acid, peptide, and protein hydroperoxides

Selenium is a critical trace element, with deficiency associated with numerous diseases including cardiovascular disease, diabetes, and cancer. Selenomethionine (SeMet; a selenium analogue of the amino acid methionine, Met) is a major form of organic selenium and an important dietary source of selenium for selenoprotein synthesis in vivo. As selenium compounds can be readily oxidized and reduced, and selenocysteine residues play a critical role in the catalytic activity of the key protective enzymes glutathione peroxidase and thioredoxin reductase, we investigated the ability of SeMet (and its sulfur analogue, Met) to scavenge hydroperoxides present on amino acids, peptides, and proteins, which are key intermediates in protein oxidation. We show that SeMet, but not Met, can remove these species both stoichiometrically and catalytically in the presence of glutathione (GSH) or a thioredoxin reductase (TrxR)/thioredoxin (Trx)/NADPH system. Reaction of the hydroperoxide with SeMet results in selenoxide formation as detected by HPLC. Recycling of the selenoxide back to SeMet occurs rapidly with GSH, TrxR/NADPH, or a complete TrxR/Trx/NADPH reducing system, with this resulting in an enhanced rate of peroxide removal. In the complete TrxR/Trx/NADPH system loss of peroxide is essentially stoichiometric with NADPH consumption, indicative of a highly efficient system. Similar reactions do not occur with Met under these conditions. Studies using murine macrophage-like J774A.1 cells demonstrate a greater peroxide-removing capacity in cells supplemented with SeMet, compared to nonsupplemented controls. Overall, these findings demonstrate that SeMet may play an important role in the catalytic removal of damaging peptide and protein oxidation products.     

Thioredoxin reductase from Plasmodium falciparum: evidence for interaction between the C-terminal cysteine residues and the active site disulfide-dithiol

Thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin by NADPH. TrxR from Plasmodium falciparum (PfTrxR) is a homodimer with a subunit Mr of 59 000. Each monomer contains one FAD and one redox active disulfide. Despite the high degress of similarity between PfTrxR and the human TrxR, their primary structures present a striking difference in the C-terminus. PfTrxR has two cysteine residues near the C-terminal Gly, while the human TrxR contains a Cys-SeCys dipeptide penultimate to the C-terminal Gly. It has been proposed that the C-terminal cysteines (as a cystine) of PfTrxR are involved in catalysis by an intramolecular dithiol-disulfide interchange with the nascent redox active dithiol. To investigate the proposed function of the C-terminal cysteines of PfTrxR, each has been changed to an alanine [Gilberger, T.-M., Bergmann, B., Walter, R. D., and Müller, S. (1998) FEBS Lett. 425, 407-410]. The single C-terminal cysteine remaining in each mutant was modified with 5,5'-dithiobis(2-nitrobenzoic acid) to form mixed disulfides consisting of the enzyme thiol and thionitrobenzoate (TNB). In reductive titrations of these mixed disulfide enzymes, 1 equiv of TNB anion was released upon reduction of the enzyme itself, while control experiments in which mutants without C-terminal cysteine were used showed little TNB anion release. This suggests that each of the C-terminal cysteines as a TNB mixed disulfide does mimic the proposed electron acceptor in the C-terminus. Analysis of the rapid reaction kinetics showed that the C-terminal mixed disulfide of the modified enzyme is reduced at a rate which is comparable with the turnover number of the wild type enzyme.     

Structural and biochemical studies reveal differences in the catalytic mechanisms of mammalian and Drosophila melanogaster thioredoxin reductases

Thioredoxin reductase (TR) from Drosophila melanogaster (DmTR) is a member of the glutathione reductase (GR) family of pyridine nucleotide disulfide oxidoreductases and catalyzes the reduction of the redox-active disulfide bond of thioredoxin. DmTR is notable for having high catalytic activity without the presence of a selenocysteine (Sec) residue (which is essential for the mammalian thioredoxin reductases). We report here the X-ray crystal structure of DmTR at 2.4 A resolution (Rwork = 19.8%, Rfree = 24.7%) in which the enzyme was truncated to remove the C-terminal tripeptide sequence Cys-Cys-Ser. We also demonstrate that tetrapeptides equivalent to the oxidized C-terminal active sites of both mouse mitochondrial TR (mTR3) and DmTR are substrates for the truncated forms of both enzymes. This truncated enzyme/peptide substrate system examines the kinetics of the ring-opening step that occurs during the enzymatic cycle of TR. The ring-opening step is 300-500-fold slower when Sec is replaced with Cys in mTR3 when using this system. Conversely, when Cys is replaced with Sec in DmTR, the rate of ring opening is only moderately increased (5-36-fold). Structures of these tetrapeptides were oriented in the active site of both enzymes using oxidized glutathione bound to GR as a template. DmTR has a more open tetrapeptide binding pocket than the mouse enzyme and accommodates the peptide Ser-Cys-Cys-Ser(ox) in a cis conformation that allows for the protonation of the leaving-group Cys by His464', which helps to explain why this TR can function without the need for Sec. In contrast, mTR3 shows a narrower pocket. One possible result of this narrower interface is that the mammalian redox-active tetrapeptide Gly-Cys-Sec-Gly may adopt a trans conformation for a better fit. This places the Sec residue farther away from the protonating histidine residue, but the lower pKa of Sec in comparison to that of Cys eliminates the need for Sec to be protonated.     

Purification and properties of a recombinant sulfur analog of murine selenium-glutathione peroxidase.

We previously constructed plasmids for synthesis of glutathione-peroxidase (GPx) mutants in an Escherichia coli expression system. In these recombinant proteins either cysteine ([Cys]GPx mutant) or serine ([Ser]GPx mutant) were present in place of the active-site selenocysteine (SeCys) of the natural enzyme. We have now investigated GPx activity of [Cys]GPx and [Ser]GPx mutants. Enzyme assays performed on preparations of these partially purified proteins demonstrated that the [Cys]GPx mutant exhibited a significant GPx activity, unlike the [Ser]GPx mutant. Purification of [Cys]GPx was performed in two steps of ion-exchange chromatography giving a 98% homogenous protein in 50% yield. The purified [Cys]GPx protein was shown to be a symmetrical tetramer by the means of gel-filtration HPLC and SDS/PAGE. Two isoelectric points were found (6.8 and 7.2) which may reflect two different oxidation states of the mutant protein. The GPx activity of the [Cys]GPx mutant was optimal at pH 8.5. The [Cys]GPx mutant had a specific activity approximately 1000-fold smaller than that of the natural enzyme, and was very easily inactivated by hydroperoxides. Inhibition of the activity with iodoacetate determined a pKa of 8.3, presumably that of the active-site cysteine. Unlike that of SeGPx, the GPx activity of [Cys]GPx was only slightly inhibited by mercaptosuccinate. We discuss hypothetical mechanistic constraints of either catalytic cycle, which may explain such results.     

Human selenium-dependent thioredoxin reductase from HeLa cells: properties of forms with differing heparin affinities.

The TrxRl form of thioredoxin reductase (TrxR) was the major form of the enzyme isolated from HeLa cells grown in a fermentor at 35 degrees C under controlled aeration conditions favorable to growth, nominally 30% of saturation of dissolved oxygen or 8 ml of oxygen in a liter of medium. This TrxR1 form was not retained on a heparin affinity matrix, it contained one selenium per subunit, was highly active catalytically, and showed strong cross-reactivity with anti-rat liver TrxR1 polyclonal antibodies. At higher aeration, 50% of saturation of dissolved oxygen or 12 ml of oxygen in a liter of medium, HeLa cell growth was slower and additional TrxR forms that bound to heparin were present in purified enzyme preparations. A minor component, TrxR2, the mitochondrial form of TrxR, was detected in the heparin-bound enzyme fraction. One enzyme form that contained less selenium (ca. 0.5 Se per TrxR subunit) was only about 50% as active with thioredoxin or 5,5'dithiobis(2-nitrobenzoic acid) as substrate. Cross-reactivity of this form with anti-rat liver TrxR1 polyclonal antibodies was very weak. The isoelectric point of the low Se enzyme, 5.85, was higher than that, 5.2-5.4, of normal Se content enzyme. Affinity of purified fully active TrxR1 to heparin could be induced by reduction with NADPH or tris-(2-carboxyethyl)phosphine (TCEP). Under anaerobic conditions there was complete retention of Se indicating that an enzyme conformation change effected by reduction was involved. The TCEP-reduced enzyme form was very oxygen labile and upon exposure to air both the Se content and catalytic activity decreased by about 50%. Addition of millimolar concentrations of NADPH or NADP(+) to the TCEP-reduced enzyme gave full protection from oxygen inactivation. TrxR1 exhibited weak peroxidase activity with H(2)O(2) as substrate in the presence of an NADPH-generating system but this activity was unstable. Specific alkylation of the selenocysteine residue of TrxR1 which completely inhibits the NADPH-dependent reduction of disulfides also destroyed peroxidase activity.     

Highly active dimeric and low-activity tetrameric forms of selenium-containing rat thioredoxin reductase 1

Mammalian thioredoxin reductase 1 (TrxR1) is a selenoprotein that contains a selenocysteine (Sec) residue at the penultimate C-terminal position. When rat TrxR1 is expressed recombinantly in Escherichia coli, partial truncation at the Sec-encoding UGA gives rise to additional protein species that lack Sec. Phenylarsine oxide (PAO) Sepharose can subsequently be used to enrich the Sec-containing protein and yield activity corresponding to that of native enzyme. Herein we extensively purified recombinant rat TrxR1 over PAO Sepharose, which gave an enzyme with about 53 U/mg specific activity. Surprisingly, only about 65% of the subunits of this TrxR1 preparation contained Sec, whereas about 35% were protein products derived from UGA truncation. Further analyses revealed a theoretical maximal specific activity of 70–80 U/mg for the homodimeric enzyme with full Sec content, i.e., significantly higher than that reported for native TrxR1. We also discovered the formation of highly stable noncovalently linked tetrameric forms of TrxR1, having full FAD content but about half the specific activity in relation to the selenium content compared to the dimeric protein. The characterization of these different forms of recombinant TrxR1 revealed that inherent turnover capacity of the enzyme must be revised, that multimeric states of the protein may be formed, and that the yield of bacterial selenoprotein production may be lower than earlier reported. The biological significance of the hitherto unsurpassed high specific activity of the enzyme involves the capacity to support a higher turnover in vivo than previously believed. The tetrameric forms of the protein could represent hitherto unknown regulatory states of the enzyme.     

Thioredoxin reductase is irreversibly modified by curcumin: a novel molecular mechanism for its anticancer activity

The thioredoxin reductase (TrxR) isoenzymes, TrxR1 in cytosol or nucleus and TrxR2 in mitochondria, are essential mammalian selenocysteine (Sec)-containing flavoenzymes with a -Gly-Cys-Sec-Gly active site. TrxRs are the only enzymes catalyzing the NADPH-dependent reduction of the active site disulfide in thioredoxins (Trxs), which play essential roles in substrate reductions, defense against oxidative stress, and redox regulation by thiol redox control. TrxRs have been found to be overexpressed by a number of human tumors. Curcumin, which is consumed daily by millions of people, is a polyphenol derived from the plant Curcuma longa. This phytochemical has well known anticancer and antiangiogenic properties. In this study we report that rat TrxR1 activity in Trx-dependent disulfide reduction was inhibited by curcumin. The IC(50) value for the enzyme was 3.6 microM after incubation at room temperature for 2 h in vitro. The inhibition occurred with enzyme only in the presence of NADPH and persisted after removal of curcumin. By using mass spectrometry and blotting analysis, we proved that this irreversible inhibition by curcumin was caused by alkylation of both residues in the catalytically active site (Cys(496)/Sec(497)) of the enzyme. However, the curcumin-modified enzyme showed a strongly induced NADPH oxidase activity to produce reactive oxygen species. Inhibition of TrxR by curcumin added to cultured HeLa cells was also observed with an IC(50) of around 15 microM. Modification of TrxR by curcumin provides a possible mechanistic explanation for its cancer preventive activity, shifting the enzyme from an antioxidant to a prooxidant.